Asymmetric partitioning of cytoplasm and generation of cellular polarity both play important roles during differentiation. We propose to further characterize genes in a system where asymmetric partitioning of organelles and cytoplasm is both obvious and amenable to genetic analyses: the amoeboid sperm of the nematode Caenorhabditis elegans. Many stages in the differentiation of this cell can be genetically dissected, its will differentiate in vitro and biochemistry on purified cells is possible. Five genes (spe-4, spe-5, spe-17, spe-16, e1947 dominant spec) with specific cytoplasmic partitioning defects during spermatogenesis will be analyzed by genetic, cytological and molecular analysis. These genes were chosen because each is associated with a mutant phenotype that affects asymmetric cytoplasmic partitioning and all have defects only in sperm. After all affect an unusual Golgi derived organelle, the fibrous body- membranous organelle (FB-MO) complexes. These organelles play a central role in partitioning of cytoplasm during the asymmetric cytokinesis that accompanies meiosis II. Depending on the mutant, disruption of FB-MO structure can either abolish the second cytokinesis or grossly affect both the cytology and function of resulting spermatids. Consequently, understanding the morphogenesis and function of the FB-MO complexes is essential in order to determine the roles that govern cellular polarity in this experimental system. The specific aims are: 1) development of a germline expression vector to allow optimized expression of reporter gene expression in the testes; 2) positional cloning of spe-16 and localizing of its encoded protein; 3) positional cloning of spe-5 and localization of its encoded protein; 4) knockout mutagenesis for a potential FB-MO resident ion channel, named ssp-20, for which we have a specific; 5) genetic analysis of dominant spe mutants have global defects in cellular polarity such that aspects of FB-MO differentiation proceed in a cell that cannot divide; 6) recovery of suppressors to begin constructing developmental pathways operative during spermatogenesis. These analysis will show how molecules involved in the sperm morphogenesis play their essential role in asymmetric cytoplasmic partitioning during sperm differentiation. This is a generally important problem during both normal cytodifferentiation and in the pathologic changes such as cancer and Alzheimer's disease. Additionally. C. elegans spermatogenesis will contribute to understanding this process in the medically and agriculturally important nematode parasites that have amoeboid sperm, but are difficult to study in the laboratory.